This invention relates to a water-insoluble tannin preparation, biologically active protein immobilized with said tannin preparation, and methods of preparation thereof.
Enzymes and other biologically active proteins isolated from tissues of living organisms (e.g., microbial cells, plants, animal tissues) have been used extensively as, for example, food additives, feed supplements, medicines, chemical reagents and industrial raw materials. Moreover, isolation and purification of proteins have been accomplished by various methods; for example, by precipitating proteins with sodium chloride, ammonium sulfate or organic solvents (e.g., acetone, ethanol) or by absorbing proteins with activated charcoal or clay. Isolation and purification may also be effected by filtering a crude protein solution through the gel of cross-linked dextran or polyacrylamide, or by column chromatography on ion exchange resins. However, the above-mentioned precipitation methods bring about co-precipitation of other high molecular compounds or inorganic materials and sometimes cause partial or total loss of the biological activity of the enzymes due to denaturation thereof. In carrying out the adsorption and chromatographic methods, various organic compounds are adsorbed, together with proteins, onto the adsorbents or ion exchange resins employed. For this reason, proteins recovered by these methods are usually contaminated with ingredients other than proteins. The gel-filtration may be useful to recover proteins in a high purity without contamination with low molecular compounds, but gel-filtration is a laborious technique and it is difficult to filter a large amount of crude protein solution through the gel.
On the other hand, immobilized enzymes (i.e., enzymes bound to carriers) have become of importance in recent years [Annual Review of Biochemistry, Vol. 35, Part II, P. D. Beyer, Editor; Annual Review Inc., Palo Alto, Calif.; pages 873-908; 1966]. Said immobilized enzymes can be used as heterogeneous catalysts in suspension or column form and, after the reactions are completed, may be readily removed from the reaction mixtures. Further, the immobilized enzymes may be used repeatedly to induce specific chemical changes in large amounts of substrate. In this connection, various methods for immobilization of enzymes have been known, including (a) covalent binding of the enzymes to suitable water-insoluble carriers such as haloacetyl-polysaccharides; (b) ionic binding to carriers such as diethylaminoethyl-(DEAE) cellulose or DEAE-Sephadex; (c) physical adsorption on inert carriers or synthetic ion exchange resins; (d) covalent cross-linking of the enzymes by bifunctional agents; (e) inclusion within the gel lattice of polyacrylamide; and (f) microencapsulation of the enzymes with semipermeable nylon membranes. Method (c) has been conducted by physically adsorbing enzymes on activated carbon or porous glass [U.S. Pat. No. 2717852, Enzymologia, 39, 12 (1970)]. These known methods (c) afford, by simple operations, the immobilized preparations having a high enzymatic activity. In said known cases, however, partial or total desorption of the enzymes is brought about even under mild conditions; for example, by changing pH or temperatures or by addition of a substrate.